Cardiac fibroblasts (CFs), along with cardiac myocytes (CMs), maintain the three-dimensional structure and electrical, chemical, and biochemical homeostasis of the myocardium. In cardiovascular disease, CF become activated, proliferate, and produce more extracellular matrix (ECM), leading to the development of fibrosis. Fibrosis can be divided in reactive (or interstitial) fibrosis, a common response to excessive myocardial loads that causes excess ECM deposition between muscle bundles, and reparative (or compact) fibrosis, a healing response to maintain structural integrity after CM death in response to injury or infarct. CFs (20% of the volume and 40-60% of the total cardiac cell population) are interwoven in densely packed CMs (75% of tissue volume but less than 50% in cells number) and can influence CMs via paracrine effects, direct cell-cell interactions, and indirect cell-ECM interactions. While regulation of CMs by CFs is known to occur in the myocardium in vivo, the mechanisms and functional significance of CF-to-CM cross talk under physiological and pathophysiological conditions are not well understood. The objective of my proposal is to determine the functional consequences and mechanisms of CF-to-CM regulation using a scaffold-free 3D model developed in my sponsor's lab that intermingles CFs and CMs as in the healthy myocardium. I will generate microtissues with interstitial fibrosis by co-seeding neonatal rat ventricular CMs with CFs that are selectively activated (Aim 1). To that end, I will utilize adenoviral gene transfer to overexpress a constitutively active mutant G?q, a key mediator of CF activation, or use CFs from rats infused with Ang II for 2 weeks in vivo. In Aim 2, I will develop 3D microtissues with activated CFs in compact configuration that separate CM mimicking compact fibrosis using individual microtissues as building blocks. In both Aims, I will test my central hypothesis is that the number of CFs, the concentration of CFs in a given area (or type of fibrosis: compact or interstitial), and the activation state of CFs influence the morphology and electrical activity in cardiac microtissues. Morphological changes of the microtissues will be characterized with histology and immunohistochemistry. Action potentials and calcium transients will be investigated as key parameters of CM function using optical mapping. Potential mechanisms by which the functional and morphological changes are driven will be investigated using immunohistochemistry, protein expression analysis, dye transfer, and gap junction inhibitors. The proposed study is expected to advance understanding of the impact of CFs on CMs the integrated functional response of cardiac microtissues and may elucidate new avenues for therapeutic strategies to treat and/or prevent structural and electrical remodeling in the diseased heart.